ISityalo sisonke se-RNA Isolation Kit Plus iyonke yeRNA Purificaiton Kit yeSityalo esiSityebi kwiiPolysaccharides kunye neePolyphenols
Inkcazelo yeKit:
Ikati.No.RE-05021/05022/05024
Ukuhlanjululwa kwe-RNA epheleleyo kwiisampuli zezityalo eziqhelekileyo eziqukethe i-polysaccharide ephezulu kunye ne-polyphenol components.
Khipha ngokukhawuleza i-RNA esemgangathweni ophezulu kwiisampuli zezityalo ezinomxholo ophezulu we-polysaccharides kunye ne-polyphenols.
RNase-Free Ukusebenzisa i-DNA-Cleaning Column
Elula-yonke imisebenzi igqitywe kwiqondo lokushisa
Ukukhawuleza-ukusebenza kunokugqitywa kwimizuzu engama-30
Ikhuselekile-akukho reagent ephilayo esetyenzisiweyo 
Iinkcukacha
50 Preps, 200 Preps
Ikiti isebenzisa ikholomu ye-spin kunye nefomula ephuhliswe yi-Foregene, enokuthi ikhuphe ngokufanelekileyo ubunyulu obuphezulu kunye nomgangatho ophezulu we-RNA kwiintlobo ezahlukeneyo zezityalo ezine-polysaccharides okanye umxholo we-polyphenols.Inika ikholomu ye-DNA-Cleaning enokuthi isuse ngokulula i-DNA ye-genomic kwi-supernatant kunye ne-tissue lysate.Ikholamu ye-RNA kuphela inokubopha ngokufanelekileyo i-RNA.Ikhithi inokuqhuba inani elikhulu leesampuli ngexesha elinye.
Inkqubo yonke ayinayo i-RNase, ngoko ke i-RNA ecocekileyo ayiyi kuthotywa.I-Buffer PRW1 kunye ne-Buffer PRW2 inokuqinisekisa ukuba i-RNA efunyenweyo ayingcoliswanga yiprotheni, i-DNA, i-ion, kunye ne-organic compounds.
Amacandelo ekhithi
| Buffer PSL1, Buffer PS, Buffer PSL2 |
| Isithinteli PRW1, Isithinteli PRW2 |
| RNase-Free ddH2O, iKholamu yokucoca iDNA |
| Ikholamu ye-RNA Kuphela |
Iimpawu&izinto eziluncedo
■ Ukusebenza kwiqondo lobushushu begumbi (15-25℃) kuyo yonke inkqubo, ngaphandle kokuhlamba komkhenkce kunye nobushushu obuphantsi be-centrifugation.
■ Gqibezela ikhithi ye-RNase-Free, akukho mfuneko yakukhathazeka malunga nokuthotywa kwe-RNA.
■ Ngokukodwa ifanele ukucocwa kwe-RNA kwiisampuli zezityalo ze-polysaccharides kunye ne-polyphenols.
∎ IKholam yokucoca i-DNA ibophelela ngokukodwa kwi-DNA, ukuze ikhithi ikwazi ukususa ungcoliseko lwe-genomic DNA ngaphandle kokongeza i-DNase.
■ Isivuno esiphezulu se-RNA: IKholamu ye-RNA kuphela kunye nefomula ekhethekileyo inokucoca ngokufanelekileyo i-RNA.
■ Isantya esikhawulezayo: kulula ukusebenza kwaye sinokugqitywa kwimizuzu engama-30.
■ Ukhuseleko: akukho sixhobo sokwenza izinto eziphilayo esifunekayo.
∎ Umgangatho ophezulu: Amaqhekeza e-RNA acociweyo anobunyulu obuphezulu, awanaprotheyini kunye nobunye ukungcola, kwaye anokumelana nemifuniselo eyahlukahlukeneyo esezantsi komlambo.
Iiparamitha zemveliso
■ Izicelo ezisezantsi: i-first-strand cDNA synthesis, RT-PCR, i-molecular cloning, i-Northern Blot, njl.
∎ Isampulu: Izihlunu zezityalo ezitsha okanye ezikhenkcezisiweyo zeepolysaccharides neepolyphenols
∎ Umthamo: 50mg izicubu zezityalo
■ Ubuninzi bomthamo wokubopha i-RNA yekholomu yokucoca: 80 μg
■ Umthamo we-Elution: 50-200 μl
Isicelo seKit
Ifanelekile ukutsalwa kunye nokucocwa kwe-RNA epheleleyo kwiisampuli zezicubu zezityalo ezitsha okanye ezikhenkcezisiweyo (ingakumbi izicubu zeqabunga lesityalo esitsha) kunye nomxholo ophezulu we-polysaccharide kunye ne-polyphenol.
Ukuhamba komsebenzi
Umzobo
Isityalo esipheleleyo se-RNA Isolation Kit Plus icutshungulwe i-50mg yamagqabi amatsha e-polysaccharides kunye ne-polyphenols, kunye ne-5% ecocekileyo ye-RNA yavavanywa yi-electrophoresis.
1: Ibhanana
2: Ginkgo
3: Umqhaphu
4: Irharnati
Ugcino kunye nobomi beShelf
Le khithi ingagcinwa iinyanga ezingama-24 phantsi kweemeko ezomileyo kumaqondo obushushu begumbi (15-25℃);ukuba ifuna ukugcinwa ixesha elide, inokugcinwa kwi-2–8℃.
I-Buffer PSL1 inokubekwa ku-4℃ kwinyanga e-1 emva kokudibanisa i-β-mercaptoethanol (kuyacetyiswa ukuba uyidibanise ngexesha elifanayo lovavanyo).
Uluhlu lufakiwe
Emva kokuba ikholamu iplagwe, isivuno se-RNA sincitshisiwe okanye akunakwenzeka ukuhlambulula i-RNA, kwaye ubunzima obufunyenweyo be-RNA buphantsi.
Uhlalutyo lwesizathu esiqhelekileyo:
1. Uqhawulo lwesampulu alucokisekanga.
Ukuqhekeka kwesampuli akubangeli ngokupheleleyo i-DNA-CLEANING COLUMN ukuba ivalwe, ngelixa ichaphazela isivuno se-RNA kunye nomgangatho.Sincoma umsebenzi wokusila ngokukhawuleza kwi-nitrogen engamanzi eyaneleyo xa uqhekeza iisampuli, Zama ukutyumza udonga lweseli yesampuli, inwebu yeseli kunye nezinye iithishu.Kwiisampuli zezityalo ze-polyol polysaccharides, sincoma ukuba usebenzise i-Plant Total RNA IOLATION KIT PLUS.
2. Xa ufunxa isampulu eyohluliweyo ye-supernatant nge-DNA-Cleaning Column, i-cell fragmented precipitate enokwenzeka inokuphefumlelwa.
Iintlenga eziqhekekileyo zeseli ezithathiweyo ziya kubangela iKholamu ye-RNA KUPHELA eya kuvalelwa xa kusenziwa umsebenzi we-adsorption we-RNA (bona inyathelo lesi-6).Siyakucebisa ngononophelo xa ufunxa le supernatant ukunqanda ubugoxo beeseli ukuba bufunxe.
3. Isixa sokuqala sesampulu sininzi kakhulu.
Ukusetyenziswa kwesampulu ngokugqithisileyo kuya kubangela ukuqhekeka kwesampulu engaphelelanga okanye i-cell lysis engaphelelanga yi-Buffer PSL1, okukhokelela ekuvalelekeni kwekholamu yokucoca ngexesha lokuhlanjululwa.I-Plant Total RNA Isolation Kit Isampuli nganye yokusebenza ecocekileyo yi-50 mg.Kwiisampulu zezityalo ze-polysaccharides ze-polyol, sincoma ukuba uzame i-Plant Total RNA IOLATION KIT PLUS.
4. Ubushushu be-centrifuge buphantsi kakhulu.
Yonke i-RNA yodwa kunye nenkqubo yokucoca iqhutyelwa kwindawo yokushisa (20-25°C), ngaphandle kokuba isampulu ithishu yaphulwe yinitrogen engamanzi.Ubushushu bezinye iicentrifuge zecryogenic bungaphantsi kwama-20.℃, enokubangela ukuvaleka kweKholamu yokucoca i-DNA kunye/okanye i-RNA-Only Column.Ukuba oku kwenzeka, setha ubushushu be-centrifuge ukuya kuma-20-25℃, kwayeqinisekisa ukuba umxube we-lysis kunye/okanye i-ethanol-eyongeziweyo ye-supernatant yayifudunyezwe ibe ngama-37°C.
Akukho RNA ekhutshiweyo okanye isivuno se-RNA siphantsi
Ngokuqhelekileyo kukho izinto ezininzi ezichaphazela ukusebenza kakuhle kokubuyisela, njenge: umxholo we-RNA yesampuli, indlela yokusebenza, umthamo we-lution, njl.
Uhlalutyo lwezizathu eziqhelekileyo zingezantsi:
I-1.I-ice bath okanye ukushisa okuphantsi (4 ° C) i-centrifugation yenziwa ngexesha lokusebenza.
Icebiso: Isebenza kwiqondo lobushushu begumbi (15-25°C) kuyo yonke inkqubo, musa ukwenza i-ice bath kunye ne-centrifugation yobushushu obuphantsi.
I-2.I-RNA iye yachithwa ngenxa yokugcinwa okungafanelekanga kwesampuli okanye ukugcinwa kwexesha elide lesampuli.
Isindululo: Iisampulu ezisandula ukuqokelelwa kufuneka zikhe zikhenkcezwe ngokukhawuleza kwinitrogen engamanzi, emva koko zigcinwe ku -80°C ixesha elide, zithintele ukuphinda kukhenkceze kunye nokunyibilika kweisampulu;okanye ngokukhawuleza ucwilise iisampuli kwi-RNA stabilizer RNAlater isisombululo (iisampulu zezilwanyana).
I-3.Ukwahlulwa kwesampulu enganelanga kunye ne-lysis kukhokelela ekuvinjweni kwekholamu yokucoca.
Isiphakamiso: Xa ugaya i-tissue, nceda uqinisekise ukuba i-tissue iphantsi ngokwaneleyo, kwaye ngokukhawuleza uyidlulisele kwi-Buffer ye-PSL1 elungiselelwe ngaphambili (qinisekisa ukuba umlinganiselo ochanekileyo we-β-ME wongezwe, jonga inyathelo 1 lenkqubo).
4.I-eluent yongezwa ngokungalunganga.
Ingcebiso: Qinisekisa ukuba i-ddH2O ye-RNase-Free ithontsizelwe embindini we-membrane yekholamu yokucoca.
5.Umthamo ochanekileyo we-ethanol epheleleyo ayizange yongezwe kwi-Buffer PSL2 okanye i-Buffer PRW2.
Ingcebiso: Nceda ulandele imiyalelo, yongeza umthamo ochanekileyo we-ethanol epheleleyo kwi-Buffer PSL2 kunye ne-Buffer PRW2 kwaye udibanise kakuhle phambi kokuba ikhithi isetyenziswe.
6.Ubungakanani besampulu yethishu ayifanelekanga.
Ingcebiso: Sebenzisa i-50 mg yethishu nge-500 μl ye-Buffer PSL1.Ukusebenzisa izicubu ezininzi kakhulu kuya kunciphisa inani le-RNA ekhutshiweyo kwaye ukucoceka kwe-RNA okubangelwayo kuya kuncitshiswa.Sincoma kakhulu ukuba idosi yesampulu yokuqala ayifanele idlule i-50 mg ngomsebenzi wokutsalwa kwe-RNA.
7.Ivolume ye-elution engafanelekanga okanye i-elution engaphelelanga.
Isiphakamiso: Umthamo ocacileyo wekholamu yokucoca ngu-50-200 μl;ukuba i-elution effect ayinelisi, kuyacetyiswa ukuba kwandiswe ixesha kwiqondo lobushushu legumbi emva kokongeza i-ddH2O ye-RNase-Free preheated, efana ne-5-10min.
I-8.Ikholamu yokucoca inentsalela ye-ethanol emva kokuhlamba nge-BufferPRW2.
Isiphakamiso: Ukuba ityhubhu engenanto i-centrifuged ye-1 min kwaye kusekho i-ethanol eseleyo emva kokuhlamba kwi-Buffer PRW2, unokwandisa ixesha le-tube engenanto ye-centrifugation ukuya kwi-2 min, okanye ubeke ikholamu yokucoca kwindawo yokushisa ye-5 min ukususa ngokupheleleyo i-ethanol eseleyo.
9.Ikhithi isetyenziswe ngokungachanekanga.
Isiphakamiso: Kwiisampuli zezityalo ze-polyphenolic polysaccharides, ukusebenzisa iikiti eziqhelekileyo ezifana ne-Plant Total RNA Isolation Kit ayinakukwazi ukufumana iisampulu ezifanelekileyo ze-RNA.Sincoma ukuba usebenzise iPlant Total RNA IsolationKit Plus, eyilelwe ngokukodwa iisampuli zezityalo ze-polyphenolic polysaccharide.Ikhithi ephuhliswe ngokukodwa ukukhupha i-RNA kwi-polyphenol kunye neesampuli zezityalo ze-polysaccharide.
Ixabiso le-OD260/OD280 liphantsi
I-RNA elution ene-ddH2O kwaye isetyenziselwa ukufundwa kwe-spectrophotometer iphumela kumaxabiso aphantsi e-OD260/OD280.Sincoma ukusebenzisa i-10 mM Tris-HCl, i-pH 7.5 (kunokuba i-RNase-Free ddH2O yokukhupha i-RNA) ukufumana amaxabiso achanekileyo e-OD260/OD280, bona “i-RNA Concentration and Purification Assays” kwiphepha le-19.
I-RNA ehlanjululweyo ithotywa
Umgangatho we-RNA ecocekileyo inxulumene nezinto ezifana nokugcinwa kwesampulu, ukungcoliseka kwe-RNase, kunye nokukhwabanisa.
Uhlalutyo lwezizathu eziqhelekileyo:
I-1.Iisampulu ze-tissue azizange zigcinwe ngexesha emva kokuqokelela.
Isincomo: Ukuba iisampulu zezicubu azisetyenziswanga ngexesha emva kokuqokelela, nceda uzigcine kwi-nitrogen engamanzi kwiqondo lokushisa eliphantsi ngokukhawuleza okanye uzidlulisele kwi--80 ° C yokugcina ixesha elide emva kokuqhwala ngokukhawuleza kwi-nitrogen engamanzi, okanye ngokukhawuleza ucwilise iisampuli kwi-RNA stabilizer RNAlater isisombululo (iisampulu zezilwanyana).Ukukhutshwa kwe-RNA, zama ukusebenzisa iisampulu zeethishu ezisanda kuqokelelwa.
2.Ukukhenkceza okuphindaphindiweyo kunye nokunyibilika kweesampuli zethishu.
Icebiso: Xa ugcina iisampulu zethishu, kungcono ukuzisika zibe ziingceba ezincinci ukuze zigcinwe, kwaye ukhuphe inxalenye yazo xa uzisebenzisa ukuthintela ukonakaliswa kweRNA okubangelwa kukukhenkcezwa okuphindaphindiweyo nokunyibilika kweisampulu.
3.I-RNase yaziswa kwigumbi lokusebenza okanye inganxitywanga iiglavu ezilahlwayo, iimaski, njl.
Ingcebiso: Imifuniselo yokutsalwa kwe-RNA yenziwa ngcono kwimisebenzi eyahlukeneyo ye-RNA, kwaye itafile yelabhoratri kufuneka icocwe phambi kovavanyo, kwaye iiglavu ezilahlwayo kunye nemaski kufuneka zinxitywe ngexesha lovavanyo ukunqanda ukuthotywa kwe-RNA okubangelwa kukwaziswa kwe-RNase ngowona mgangatho mkhulu.
I-4.I-reagent ingcolisekile yi-RNase ngexesha lokusetyenziswa.
Ingcebiso: Faka endaweni yoluhlu olutsha lwezixhobo ze-RNA zokutsalwa kwezityalo zizonke zemifuniselo enxulumeneyo.
I-5.Iibhubhu ze-centrifuge kunye neengcebiso ze-pipette ezisetyenziselwa ukuguqulwa kwe-RNA zingcolile nge-RNase.
Ingcebiso: Qinisekisa ukuba iityhubhu ze-centrifuge, iingcebiso ze-pipette, ii-pipettes, njl njl. ezisetyenziswa kwi-RNA extraction zonke zi-RNase-Free.
Izikhokelo zoMyalelo:
Plant Total RNA Isolation Kit Plus Instruction Manual
